Millions and millions of people have been infected with the human immunodeficiency virus (“HIV”), the causative agent of acquired immune deficiency syndrome (“AIDS”), since the early 1980s. HIV/AIDS is now the leading cause of death in sub-Saharan Africa, and is the fourth biggest killer worldwide. At the end of 2001, an estimated 40 million people were living with HIV globally.
Currently, five classes of antiretroviral drugs are used to treat infection by Human Immunodeficiency Virus (HIV), i.e. protease inhibitors (PIs), two classes of reverse transcriptase inhibitors (nucleoside reverse transcriptase inhibitors abbreviated as N RTI and non-nucleoside reverse transcriptase inhibitors abbreviated as NN-RTI), entry inhibitors (fusion inhibitors (FIs) and co-receptor antagonists), and integrase inhibitors (INIs). Integrase inhibitors are a promising new class of antiretrovirals interfering with HIV replication by blocking the ability of the virus to integrate into the genetic material of human cells.
Modern anti-HIV drugs target different stages of the HIV life cycle and a variety of enzymes essential for HIV's replication and/or survival. Amongst the drugs that have so far been approved for AIDS therapy are nucleoside reverse transcriptase inhibitors (“NRTIs”) such as AZT, ddl, ddC, d4T, 3TC, and abacavir; nucleotide reverse transcriptase inhibitors such as tenofovir; non-nucleoside reverse transcriptase inhibitors (“NNRTIs”) such as nevirapine, efavirenz, and delavirdine; protease inhibitors (“PIs”) such as darunavir, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir and atazanavir; fusion inhibitors, such as enfuvirtide, co-receptor antagonists such as maraviroc and integrase inhibitors such as raltegravir.
Nonetheless, in the vast majority of subjects none of the antiviral drugs currently approved, either alone or in combination, proves effective either to prevent eventual progression of chronic HIV infection to AIDS or to treat acute AIDS. This phenomenon is due, in part, to the high mutation rate of HIV and the rapid emergence of mutant HIV that are resistant to antiviral therapeutics upon administration of such drugs to infected individuals.
The integrase protein thus represents an interesting target for HIV inhibitor research. HIV integrase is required for integration of the viral genome into the genome of the host cell, a step in the replicative cycle of the virus. HIV integrase is a protein of about 32 KDa encoded by the pol gene, and is produced in vivo by protease cleavage of the gag-pol precursor protein during the production of viral particles. The integration process takes place following reverse transcription of the viral RNA. First, the viral integrase binds to the viral DNA and removes two nucleotides from the 3′ end of the viral long-terminal repeat (LTR) sequences on each strand. This step is called 3′ end processing and occurs in the cytoplasm within a nucleoprotein complex termed the pre-integration complex (PIC). Second, in a process called strand transfer, the two strands of the cellular DNA into which the viral DNA will be inserted, the target DNA, is cleaved in a staggered fashion. The 3′ ends of the viral DNA are ligated to the 5′ ends of the cleaved target DNA. Finally, host enzymes probably repair remaining gaps.
With the increasing number of available anti-HIV compounds as mentioned above, the number of potential treatment protocols for HIV infected patients will continue to increase. Many of the currently available compounds are administered as part of a combination therapy. The high complexity of treatment options coupled with the ability of the virus to develop resistance to HIV inhibitors requires the frequent assessment of treatment strategies. The ability to accurately monitor the replicative capacity of virus in patients with a drug regimen and to use that data to modify the doses or combinations of inhibitors allows physicians to effectively reduce the formation of drug resistant virus and provide an optimal, tailored treatment for each patient.
Accordingly, as new drugs targeting new HIV polypeptides become available, phenotypic and genotypic assays for determining resistance or susceptibility of HIV infecting a patient to such new anti-HIV drugs are highly needed.
While phenotyping and genotyping assays have been developed and marketed for reverse transcriptase and protease genes, protocols and assays for evaluation of drug resistance against the integrase gene have not been successfully developed.
For instance, the amplicon used in the marketed Antivirogram® contains the gag cleavage sites (p1/p7 and p1/p6), PR (codon 1-99) and RT (codon 1-400) coding sequences respectively, leaving the rest of the relevant HIV reverse transcriptase gene and more importantly the HIV integrase gene undetected.